title: Codon optimization, expression, purification, and functional characterization of recombinant human IL-25 in Pichia pastoris.
abstract: Interleukin (IL)-25 (also known as IL-17E) is a distinct member of the IL-17 cytokine family which induces IL-4, IL-5, and IL-13 expression and promotes pathogenic T helper (Th)-2 cell responses in various organs. IL-25 has been shown to have crucial role between innate and adaptive immunity and also a key component of the protection of gastrointestinal helminthes. In this study, to produce bioactive recombinant human IL-25 (rhIL-25), the cDNA of mature IL-25 was performed codon optimization based on methylotropic yeast Pichia pastoris codon bias and cloned into the expression vector pPICZαA. The recombinant vector was transformed into P. pichia strain X-33 and selected by zeocin resistance. Benchtop fermentation and simple purification strategy were established to purify the rhIL-25 with about 17 kDa molecular mass. Functional analysis showed that purified rhIL-25 specifically bond to receptor IL-17BR and induce G-CSF production in vitro. Further annexin V-FITC/PI staining assay indicated that rhIL-25 induced apoptosis in two breast cancer cells, MDA-MB-231 and HBL-100. This study provides a new strategy for the large-scale production of bioactive IL-25 for biological and therapeutic applications.
authors.affiliation: Key Laboratory of Molecular Medicine, Ministry of Education, Department of Biochemistry and Molecular Biology, Shanghai Medical College, Fudan University, Room 111, Building 13, No. 130 Dong'an Road, Xuhui District, Shanghai, 200032, People's Republic of China.
authors.forename: Yushan
authors.initials: Y
authors.lastname: Liu
authors.forename: Chengsheng
authors.initials: C
authors.lastname: Wu
authors.forename: Jinyu
authors.initials: J
authors.lastname: Wang
authors.forename: Wei
authors.initials: W
authors.lastname: Mo
authors.forename: Min
authors.initials: M
authors.lastname: Yu
mesh_terms: D017209:Apoptosis; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D003062:Codon; D015870:Gene Expression; D016179:Granulocyte Colony-Stimulating Factor / Q000378:metabolism; D006801:Humans; D020381:Interleukin-17 / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000235:genetics; D011485:Protein Binding; D053722:Receptors, Interleukin-17 / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D017422:Sequence Analysis, DNA
chemical_list: D003062:Codon; C486701:IL25 protein, human; D020381:Interleukin-17; D053722:Receptors, Interleukin-17; D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor